The long-term objective is to determine the RNA sequence and structural elements in a ribonuclease III processing substrate that direct specific binding of enzyme and promote processing catalysis. The ribonuclease III substrate is the bacteriophage T7 R1.1 processing signal, an approximately 60 nucleotide RNA hairpin contained in the T7 early mRNA precursor. The R1.I substrate undergoes cleavage by RNase III at a single specific site in vitro, with gene regulatory consequences. The specific aims are to: . Identify the nucleotides that directly participate in binding RNase III, through chemical and enzymatic probing of the RNase III-R1.1 RNA complex. . Identify the RNA base-pairs and functional groups that form the RNase III recognition elements, through analyzing the processing reactivities and binding affinities of R1.1 sequence variants. . Determine the involvement of magnesium ion in substrate recognition by employing an RNase III point mutant (Rnc70) which binds substrate, but is incapable of Mg2+-catalyzed cleavage. Ongoing collaborative proton NMR analysis of the R1.1 processing signal and an x-ray crystallographic study of RNase III and its complex with substrate will provide important structural information to complement the biochemical studies. This project has additional important implications regarding human health and disease. Specifically, RNase III has been shown to have strong structural and/or functional similarities to other dsRNA-binding proteins, including a mammalian nuclear protein that binds the human immunodeficiency virus (HIV) TAR element, and a mammalian dsRNase that is induced by interferon as part of the antiviral response. Understanding RNase III processing catalysis can provide a basis to define the role of protein-dsRNA interactions in human health and disease.